CONVERGENCE OF TCDD GENE ACTIVATION WITH OTHER PATHWAYS

描述（摘自申请人摘要）：Ah受体（AHR）是一种配体

DESCRIPTION (from Applicant's Abstract): The Ah receptor (AHR) is a ligand activated transcription factor that binds ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and is thought to mediate many of the highly toxic and carcinogenic actions of TCDD. The AHR and its DNA binding partner ARNT (Ah receptor nuclear translocator) recognize specific DNA sequences, referred to as dioxin responsive element (DREs) resulting in the activation of gene transcription. Both the AHR and ARNT are basic/helix-loop-helix proteins. Basic/helix-loop-helix proteins are characterized by their ability to form homo- or hetero-dimers, contact DNA via their basic domains and form a variety of DNA binding species that recognize distinct DNA sequences and regulate specific sets of genes. Contrary to the generally accepted theory that TCDD exerts its adverse affects through the regulation of DRE responsive genes, we propose that TCDD actions include nuclear localization of the AHR, dimerization of ARNT and the subsequent removal of ARNT from the regulatory regions of other genes. This hypothesis suggests that ARNT, in the absence of the AHR regulates genes and that this regulation is adversely affected by the presence of TCDD and the AHR. Our preliminary data has shown that ARNT can form multiple partnerships. The ARNT homodimer recognizes the CACGTG sequence, the recognization site of the Myc/Max heterodimer involved in regulating cellular proliferation and differentiation. We will use Myc/Max gene regulation as a model of AHR-independent ARNT gene regulation. First, we will determine whether the ARNT homodimer will recognize the CACGTG site with high affinity required for biological significance and compare this binding affinity to that of Myc and Max. Then we will use transient transfection assays to determine whether theARNT homodimer formation may repress biological processes such as the induction of cytochrome P4501A1 and Myc induced cellular transformation. To determine why the AHR ARNT recognize different DNA half-sites, we will perform site-directed mutagenesis of their DNA binding regions followed by footprinting analysis. Finally, we will use cell-free transcription to identify the interacting transcription factors that facilitate AHR and ARNT gene regulation and determine whether different AHR and ARNT ratios found in vivo may affect the ability of the AHR and ARNT to heterodimerize and resulting differential gene regulation. The broad, long-term objectives of this proposal are to understand how the AHR and ARNT regulate and the mechanisms that dictate the tissue-specific effects of TCDD toxicity and carcinogenicity.